This invention pertains to assays for the detection of particular target nucleic acid sequences and specific protein, carbohydrate, steroid and hapten analytes in a sample to be inspected. This application is assigned to Tropix, Inc., a leader in chemiluminescent assays, and reagents therefor. The common assignee has pioneered the development and patenting of 1,2-dioxetane chemiluminescent substrates for use in the detection of target analytes of a sample, including immunological assays of a wide variety. While the list of patents is exhaustive, prominent patents include U.S. Pat. Nos. 5,112,960 and 5,538,847, which disclose dioxetanes particularly adapted for improved assay properties, as well as U.S. Pat. Nos. 5,145,772 and 4,978,614 which disclose methods of enhancing the intensity of chemiluminescence obtained from these chemiluminescent molecules. As broadly described, these dioxetane molecules have the structure set forth below.
In these 1,2-dioxetanes, T is a stabilizing group. Because the dioxetane molecule, without the stabilizing group, may spontaneously decompose, a group, typically a polycycloalkyl group is bound to the dioxetane to stabilize it against spontaneous decomposition. This need for stabilization has resulted in commercially developed 1,2-dioxetanes being generally spiroadamantyl. The adamantyl group, spiro-bound, can be substituted at any bridge head carbon, to affect chemiluminescent properties. As indicated, the remaining carbon of the dioxetane ring bears a OR substituent, wherein R is generally an alkyl or cycloalkyl, although it may be a further aryl group. Preferred embodiments include substituted alkyls, with the substituent including halogenated groups, such as polyhaloalkyl substituents. The remaining valence is occupied by an aryl moiety, preferably phenyl or naphthyl. If naphthyl, particular substitution profiles on the naphthyl ring are preferred. See, e.g., U.S. Pat. No. 4,952,707. The aryl ring bears at least one substituent, X. In commercially developed dioxetanes, this is an enzyme-cleavable group. For instance, many assays employ an exogenous enzyme, such as alkaline phosphatase, to ensure reliability of the assay. The enzyme is typically conjugated to a binding ligand, either an antibody, a nucleic acid fragment, or similar binding pair member, which will bind to the target substance to be detected. Where the conjugated enzyme is alkaline phosphatase, the enzyme-cleavable group X will be a phosphate. The aryl ring may also bear a substituent Y, which is selected to be either electron donating, or electron withdrawing. Preferred groups include chlorine, alkoxy and heteroaryl, although other groups may be employed. These substitutions further effect chemiluminescent properties, and reaction kinetics. A wide variety of other substituents are disclosed in the afore-referenced patents, all of which are incorporated herein by reference.
Uniformly, these dioxetanes are disclosed as useful enzyme substrates, that is, the binding pair member conjugated to an enzyme is allowed to bind to the target analyte, and after washing to remove unbound material, the dioxetane is added. In the presence of the conjugated enzyme, the protective group is cleaved, leading to decomposition of the dioxetane, and light emission. These dioxetanes provide a variety of valuable properties for the detection of biomolecules that had not been previously available. These include detection sensitivity, thermal stability, water solubility and ease of use. Employing the enzyme amplification factor, the dioxetanes commercially available offer the highest detection sensitivity attainable in commercialized assays. Similarly, the thermal stability of the described dioxetanes is superior to that of radioisotopes, fluorophores and other available chemiluminescent systems. Because biological assay conditions generally employ an aqueous media, water solubility, an important criteria, was met by use of the dioxetane substrates, which proved easy to use in both qualitative and quantitative determinations, in solutions, and in blotting assays.
Increasingly, however, PCR-amplified probe hybridization assays, or in-situ applications, are receiving commercial attention. Enzyme labels, a prerequisite for use of the enzyme-cleavable dioxetanes described, may not always be appropriate for such assays. For example, a short oligonucleotide probe used to detect a specific target nucleic acid sequence is difficult to label with an enzyme. Most enzymes have high molecular weight, are large and may easily interfere-with the hybridization of the labeled probe to a target DNA sequence. Further, most enzymes cannot be subjected to the harsh conditions used in processing nucleic acids, such as high temperatures and organic or inorganic solvents.
Further, a wide variety of assays, including in-situ nucleic acid detection, chromosome analysis, protein detection, such as cell-surface antigens and related applications require microscopic analysis, demanding very high resolution capability. Further, the trend in detection methodologies appears to be moving toward miniaturization and high density in order to achieve efficiency, including the use of electromagnetically sensitive surfaces such as semiconductors and microchips. See, e.g., Eggers et al, 516 BioTechniques, Vol. 17, No. 3 (1994). For these applications, there is a need for light emitters, or other labels, which produce points of light at a very precise location on a microscopic level. This implies a need for immobilized labels which emit light while covalently attached, or otherwise positively bound, to a ligand or binding agent. In contrast, dioxetane enzyme substrates are added to bound enzyme labeled reagents, and are activated by the removal of the cleavable group by the enzyme, which leaves an oxyanion intermediate, which may diffuse randomly, until decomposition, and chemiluminescence, is achieved. While the hydrophobic affinity of the environment may passively “bind” the dioxetane anion, the lack of an attachment of the anion to the probe allows limited diffusion, which limits imaging on a microscopic scale.
Moreover, the high sensitivity required for many immunological assays, met by the enzyme-cleavable dioxetanes described above, may not be necessary with PCR. PCR, and linear target amplification techniques including cycling pre technology (CPT), and oligonucleotide ligase assay (OLA), appear to be the future technology of choice for many nucleic acid detection formats. Since these techniques all offer some degree of amplification, with that of PCR being generally considered the greatest, ease of use, potential for an homogenous assay and universality may be features that are selected over label sensitivity.
Further, the use of nucleic acid probes and the like for assays implies a set of conditions that may be unlike those for which the enzyme-cleavable dioxetanes were originally developed. Thus, water solubility may not be required. For chemiluminescently labeled detection of nucleic acids, water solubility is not critical. A partial or total use of solvents is not only possible, but it may dramatically enhance the chemiluminescent efficiency of the label (it is well established that in many organic solvents, chemiluminescent efficiency of the aryl-substituted 1,2-dioxetane decomposition is improved).
Thus, there remains a need in the industry to develop chemiluminescent dioxetane labels for nucleic acid probes and related technologies that can provide high signal resolution, resistance to hybridization conditions, and reliable emission properties as labels in a nucleic acid assay.